The Glycoprotein Hormone Family
The glycoprotein hormone family contains three members, namely lutropin (LH, which is also known a luteinizing hormone or interstitial cell stimulating hormone), follitropin (FSH, which is also known as follicle stimulating hormone), and thyrotropin (TSH, which is also known as thyroid stimulating hormone). Lutropins and follitropins of fish are also known as gonadotropin II (GTHII) and gonadotropin I (GTHI), respectively. These glycoprotein hormones are made in the anterior pituitary gland. The placentas of humans, other primates, and some mammals—e.g., horses—also make a glycoprotein hormone known as choriogonadotropin (CG) that has a similar or identical amino acid sequence as that of lutropin. CG interacts with lutropin receptors and, in some cases—e.g., equine CG—can interact with lutropin and follitropin receptors of many species (Murphy and Martinuk, 1991). CG has a role in the maintenance of pregnancy and that of humans (hCG) is essential for maintaining early pregnancy. Its presence in the urine of pregnant women is the basis of most pregnancy tests. hCG is also produced by many tumors and its presence in men and non-pregnant women is an indication of malignancy.
α and β Subunits
Glycoprotein hormones are composed of two subunits termed α and β (Pierce and Parsons, 1981). A single gene encodes the α-subunit in most vertebrate species and this subunit is common to lutropins, follitropins, thyrotropins, and choriogonadotropins. Post-translational modifications of the glycoprotein hormones can create differences in their α-subunits such as the finding that the α-subunit of LH usually contains a higher ratio of sulfate to sialic acid than that of FSH (Baenziger and Green, 1988). Some fish have two α-subunit genes that encode sequences that differ primarily in loops α1 and α3. The β-subunits of lutropins, follitropins, and thyrotropins are encoded by separate genes. Similarities in the locations of the cysteines and other residues in the β-subunits of all glycoprotein hormones suggest that genes encoding the β-subunits arose by gene duplication and then diverged during early vertebrate evolution (Li and Ford, 1998). The evolution of the primate CG genes occurred much later, most likely by read-through and duplication of the LH gene (Fiddes and Talmadge, 1984). Although the β-subunit controls the biological properties of each hormone (Pierce and Parsons, 1981), both hormone subunits are required for full activity in most assays.
Heterodimer Formation
Heterodimer formation and dissociation in vitro requires that the glycosylated end of α2 pass beneath the seatbelt through a hole in the β-subunit. The seatbelt presents a significant impediment to heterodimer dissociation and assembly at physiological temperature and pH (Xing et al., 2001b). This is due largely to the presence of the oligosaccharide on α2 as shown by the fact that its removal facilitates assembly (Xing and Moyle, 2003), a phenomenon that can be used as a method for preparing heterodimers lacking this oligosaccharide. Normally, the heterodimer is stable at pH 3-4 and above. Removal of the α−2 oligosaccharide decreases heterodimer stability significantly and, with the exception of heterodimers in which the seatbelt is latched to a cysteine in the aminoterminal end of the β-subunit, the absence of the α2 oligosaccharide renders the heterodimer unstable at pH 5. Heterodimers in which the seatbelt is latched to a cysteine in the aminoterminal end of the β-subunit are usually much more stable than those in which the seatbelt is latched to a cysteine in β-subunit loop 1.
Due to the role of the seatbelt in heterodimer stability, it was thought that the heterodimer was assembled before the seatbelt became latched. This notion was supported by studies using pulse chase analyses (Ruddon et al., 1996). Extensive studies of heterodimer formation in the endoplasmic reticulum (Xing et al., 2004a; Xing et al., 2004b; Xing et al., 2004c; Xing et al., 2004d), the major site of glycoprotein hormone assembly, revealed that it occurs by two mechanisms (FIG. 3). In one termed the “wraparound” pathway, the subunits dock before the seatbelt is latched, the seatbelt is wrapped around α2, and assembly is completed when the seatbelt becomes latched. Although this process is required for assembly of salmon FSH and other piscine follitropins in which the seatbelt is latched to a cysteine in the aminoterminal region of the β-subunit (Xing et al., 2004c), it is inefficient for at least two reasons. First, the β-subunit has a tendency to fold completely prior to heterodimer assembly unless it is prevented from doing so by the composition of the seatbelt or by a chaperone that interferes with seatbelt latching. Mammalian cells have a chaperone that impedes latching of the human LH seatbelt before the subunits dock, a phenomenon that facilitates the assembly of human LH by the wraparound pathway. The second impediment to assembly by the wraparound pathway stems from the fact that the unlatched seatbelt destabilizes the transient complex composed of the α-subunit and the unlatched β-subunit (Xing et al., 2004d). These factors appear to be largely responsible for the difficulty of producing salmon FSH and many other piscine follitropins that have similar folding patterns. Since FSH is required for producing the female gametes of all vertebrates, methods that are capable of overcoming this difficulty of assembly or that are capable of producing active follitropin analogs would be desirable.
The heterodimer can also be assembled by a “threading pathway” in which the glycosylated end of α-subunit loop 2 passes beneath the seatbelt. This process is facilitated substantially by the presence of small concentrations of reducing agents (Xing et al., 2001b). Originally, it was thought that reduction disrupted the seatbelt latch disulfide, which enabled the heterodimer to form by the wraparound pathway. Reducing agents are now known to enhance assembly by disrupting the disulfide that stabilizes the small loop in the aminoterminal half of the seatbelt (Xing et al., 2001b). The redox potential of the endoplasmic reticulum promotes disruption of this disulfide in cells during the assembly of most choriogonadotropins, follitropins, and thyrotropins (Xing et al., 2004a). The ability of 1-3 mM (3-mercaptoethanol to promote assembly in vitro is due to the fact that the disulfide that stabilizes the small seatbelt loop is much more stable in the heterodimer than in the free β-subunit. Its stability in the heterodimer is due largely to interactions between the α- and β-subunits that stabilize the positions of β-subunit cysteines 10 and 11 near one another (FIG. 3). Disruption of the disulfide formed by these cysteines lengthens the seatbelt, which facilitates the passage of the glycosylated end of α2 between the seatbelt and the remainder of the β-subunit. This process, termed “threading” (Xing et al., 2001b; Xing et al., 2004a; Xing et al., 2004b; Xing et al., 2004d), is driven by the formation of a hydrogen bond network between α2 and the β-subunit that drags the glycosylated end of α2 beneath the seatbelt (FIG. 3, lower pathway). Once threading is complete, the proximity of α2 to the residues that form the small seatbelt loop promotes reformation of the disulfide that stabilizes the small seatbelt loop and that stabilizes the heterodimer (Xing et al., 2004a; Xing et al., 2004b; Xing et al., 2004d). This is why concentrations of reducing agents that are sufficient to promote assembly do not cause heterodimer dissociation. Due to the fact that the disruption and formation of the small seatbelt loop lengthens and shortens the seatbelt, this loop can be viewed as a “tensor” and the disulfide that stabilizes this loop can be viewed as “the tensor disulfide” (Xing et al., 2004a; Xing et al., 2004b; Xing et al., 2004d). Threading promotes the efficient assembly of heterodimers such as hCG, hFSH, and hTSH in which the seatbelt is latched to a cysteine in β1. It appears unable to facilitate assembly of heterodimers in which the seatbelt is latched to a cysteine in the aminoterminal end of the β-subunit—e.g., salmon FSH.
Receptor Binding Specificity
In addition to its role in stabilizing the heterodimer, the seatbelt has a substantial influence on receptor binding specificity. Indeed, the seatbelt is responsible for much of the influence of the hormone β-subunit on receptor binding specificity (Moyle et al., 1994; Han et al., 1996; Dias et al., 1994; Grossmann et al., 1997). Remarkably, the aminoterminal and carboxyterminal halves of the seatbelt appear to have separate influences on receptor binding specificity. The aminoterminal half has a much greater influence on binding to LH receptors than the carboxyterminal half. Conversely, the carboxyterminal half of the seatbelt has a much greater influence on binding to FSH receptors than the aminoterminal half. By changing the composition of the seatbelt, one can produce hormone analogs that interact with multiple receptors. Replacing hCG β-subunit residues between cysteines 11 and 12 with their FSH β-subunit counterparts led to a hormone analog that had the same high affinity for LH receptors as hCG and about half the affinity of FSH receptors for FSH (Moyle et al., 1994). By manipulating the composition of the seatbelt loop in this analog, one can alter the ratio of LH/FSH activity more than 100-fold (Han et al., 1996).
Glycoprotein Hormone Agonists and Antagonists
Efforts to design glycoprotein hormone agonists and antagonists would be facilitated by knowledge of the structures of their receptors and these membrane glycoproteins have been studied extensively. Receptors for all three hormone classes have similar components, namely a large extracellular domain, a rhodopsin-like (Palczewski et al., 2000) transmembrane domain (TMD), and a short cytoplasmic carboxyterminal domain. The cytoplasmic carboxyterminus is not needed for receptor expression or signaling (Sanchez-Yague et al., 1992; Zhu et al., 1993). The extracellular domain contains two subdomains. The largest of these contains approximately 250 residues and is composed of leucine-rich repeats (McFarland et al., 1989; Sprengel et al., 1990; Nagayama et al., 1989). The leucine-rich repeat domain (LRD) was modeled several years ago (Moyle et al., 1995; Kajava et al., 1995; Jiang et al., 1995) based on its similarity to ribonuclease inhibitor, the first leucine-rich repeat protein of known structure (Kobe and Deisenhofer, 1993). The LRD creates at least a part of the ligand binding site (Braun et al., 1991) and a crystal structure of hFSH bound to a fragment of the LRD has been determined (Fan and Hendrickson, 2005), although there is some doubt as to relevance of this structure to the manner in which the glycoprotein hormone ligands dock with their cell surface receptors (Moyle et al., 2005). Depending on the receptor, the remainder of the extracellular domain contains approximately 60-150 residues. This portion of the extracellular domain has been more difficult to model, however, since its amino acid sequence is not similar to proteins of known structure. It connects the LRD to the TMD and is often considered a hinge (Jiang et al., 1995; Ji et al., 2002; Rapoport et al., 1998; Dias, 2005; Fan and Hendrickson, 2005) and many diagrams suggest that it is disordered (FIG. 4). It has also been termed the signaling-specificity domain (SSD) to reflect its roles in ligand binding and signal transduction (Moyle et al., 2004). The SSD may make essential contacts with the LRD and TMD (FIG. 5a,b), a phenomenon that would permit the receptor domains to function as an integrated unit. The SSD—i.e., the “hinge region”—is considered to be highly ordered in these models.
Models for Hormone-Receptor Interactions
Two models have been proposed to explain hormone receptor interactions. That favored by most investigators was devised several years ago (Jiang et al., 1995) and is supported by the crystal structure of hFSH bound to a fragment in the human FSH receptor (Fan and Hendrickson, 2005). In the crystal structure hFSH is oriented perpendicular to the concave surface of the LRD (FIGS. 4 and 6), an orientation proposed to explain binding of all glycoprotein hormones to their receptors (Fan and Hendrickson, 2005). In this model the role of the SSD is merely to link the LRD to the TMD in a fashion that permits bound ligand to contact the extracellular loops of the TMD (Fan and Hendrickson, 2005; Remy et al., 1996; Dias, 2005). This widely perceived model served as the logo for the latest international meeting of glycoprotein hormone biologists that was held Apr. 13-17, 2005 (FIG. 5a). Signal transduction is thought to be initiated by dimerization of the LRD through contacts between its convex surface (FIG. 7).
A contrasting view of the receptor (Moyle et al., 2004) maintains that ligands contact the glycosylated surfaces of the LRD and SSD, not the TMD or the concave surface of the LRD as is seen in the crystal structure (Fan and Hendrickson, 2005). Indeed, since the SSD would block access of the ligand to the concave surface of the LRD, both models of ligand binding are mutually exclusive. In the alternate view of the receptor (FIG. 5), the SSD has a compact shape and does not function as a hinge. It has been modeled on the structure of the KH domain (Moyle et al., 2004) and aligned with the concave surface of the LRD and TMD (FIG. 5). Signal transduction depends on interactions between the LRD, SSD, and TMD. Although the LRD has an important role in ligand binding affinity and specificity (Moyle et al., 1994; Segaloff and Ascoli, 1993; Nagayama et al., 1991; Thomas et al., 1996; Xie et al., 1990; Braun et al., 1991), interactions between all three domains contribute to ligand binding specificity and signaling. This explains why the LRD is not the only part of the receptor known to influence binding of most ligands (Abell et al., 1996; Moyle et al., 1994; Bernard et al., 1998; Nagayama et al., 1991; Moyle et al., 2004).
Problems in Attempts to Design Agonists and Antagonists
The lack of structural knowledge has hampered development of glycoprotein hormone agonists and antagonists. Although methods for producing glycoprotein hormones were developed in 1985 (Reddy et al., 1985), these are not applicable to all ligands. For example, it has been particularly difficult to produce hormones and hormone analogs in which the seatbelt is latched to a cysteine in the N-terminal region of the β-subunit. This has limited the development of analogs that can be used to stimulate fertility in not only mammalian systems but also those of salmon, trout, and other fish that express follitropins (in which the seatbelt is latched to a cysteine in the aminoterminal region of the β-subunit).